Practical use of ethyl glucuronide and ethyl sulfate in postmortem cases as markers of antemortem alcohol ingestion

In postmortem toxicology, it could be difficult to determine whether a positive blood ethanol concentration reflects antemortem ingestion or postmortem synthesis of alcohol. Measurement of the nonoxidative ethanol metabolite ethyl glucuronide (EtG) has been suggested as a marker of antemortem ingestion of alcohol, but EtG might degrade postmortem which could make interpretation difficult. So far, the published articles concern EtG only. Another nonoxidative metabolite, ethyl sulfate (EtS), which is more stable, has therefore been included in this study. We present a material of 36 deaths where postmortem formation of ethanol was suspected and where both EtG and EtS were measured in blood and urine to assist the interpretation. In 19 cases, EtG and EtS were positive in the body fluids analyzed. The median concentration of EtG and EtS in blood was 0.4 (range 0.1–23.2) and 0.9 mg/L (range 0.04–7.9), respectively. The median concentration of EtG and EtS in urine was 35.9 (range 1.0–182) and 8.5 mg/L (range 0.3–99), respectively. In another 16 cases, there was no trace of EtG or EtS in the specimens analyzed. In one case, there was inconsistency between the results of EtG and EtS; they were both positive in urine, while only EtS was positive in blood. This study showed that, out of 36 cases, antemortem ingestion of alcohol was very likely in 19 and unlikely in 16, according to EtG and EtS results. In the last case, the interpretation was more difficult. One possible explanation would be postmortem degradation of EtG in blood.     

Assistance of ethyl glucuronide and ethyl sulfate in the interpretation of postmortem ethanol findings

Postmortem ethanol formation is a well-known problem in forensic toxicology. The aim of this study was to interpret findings of ethanol in blood, in a large collection of forensic autopsy cases, by use of the nonoxidative ethanol metabolites, ethyl glucuronide (EtG), and ethyl sulfate (EtS). In this study, according to previously published literature, antemortem ethanol ingestion was excluded in EtS-negative cases. Among 493 ethanol-positive forensic autopsy cases, collected during the study period, EtS was not detected in 60 (12 %) of the cases. Among cases with a blood alcohol concentration (BAC) of ≤0.54 g/kg, antemortem ethanol ingestion was excluded in 38 % of the cases, while among cases with a BAC of ≥0.55 g/kg, antemortem ethanol ingestion was excluded in 2.2 % of the cases. For all cases where ethanol was measured at a concentration >1.0 g/kg, EtS was detected. The highest blood ethanol concentration in which EtS was not detected was 1.0 g/kg. The median concentrations of EtG and EtS in blood were 9.5 μmol/L (range: not detected (n.d.) 618.1) and 9.2 μmol/L (range: n.d. 182.5), respectively. There was a statistically significant positive correlation between concentration levels of ethanol and of EtG (Spearman’s rho?=?0.671, p?p?相似文献   

State-of-the-art of bone marrow analysis in forensic toxicology: a review

Although blood is the reference medium in the field of forensic toxicology, alternative matrices are required in case of limited, unavailable or unusable blood samples. The present review investigated the suitability of bone marrow (BM) as an alternative matrix to characterize xenobiotic consumption and its influence on the occurrence of death. Basic data on BM physiology are reported in order to highlight the specificities of this matrix and their analytical and toxicokinetic consequences. A review of case reports, animal and human studies involving BM sample analysis focuses on the various parameters of interpretation of toxicological results: analytic limits, sampling location, pharmacokinetics, blood/BM concentration correlation, stability and postmortem redistribution. Tables summarizing the analytical conditions and quantification of 45 compounds from BM samples provide a useful tool for toxicologists. A specific section devoted to ethanol shows that, despite successful quantification, interpretation is highly dependent on postmortem interval. In conclusion, BM is an interesting alternative matrix, and further experimental data and validated assays are required to confirm its great potential relevance in forensic toxicology.     

Determination of ethyl glucuronide and ethyl sulfate from dried blood spots

Background

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are non-oxidative minor metabolites of ethanol. They are detectable in various body fluids shortly after initial consumption of ethanol and have a longer detection time frame than the parent compound. They are regarded highly sensitive and specific markers of recent alcohol uptake. This study evaluates the determination of EtG and EtS from dried blood spots (DBS), a simple and cost-effective sampling method that would shorten the time gap between offense and blood sampling and lead to a better reflectance of the actual impairment.

Methods

For method validation, EtG and EtS standard and quality control samples were prepared in fresh human heparinized blood and spotted on DBS cards, then extracted and measured by an LC-ESI-MS/MS method. Additionally, 76 heparinized blood samples from traffic offense cases were analyzed for EtG and EtS as whole blood and as DBS specimens. The results from these measurements were then compared by calculating the respective mean values, by a matched-paired t test, by a Wilcoxon test, and by Bland–Altman and Mountain plots.

Results and discussion

Calibrations for EtG and EtS in DBS were linear over the studied calibration range. The precision and accuracy of the method met the requirements of the validation guidelines that were employed in the study. The stability of the biomarkers stored as DBS was demonstrated under different storage conditions. The t test showed no significant difference between whole blood and DBS in the determination of EtG and EtS. In addition, the Bland–Altman analysis and Mountain plot confirmed that the concentration differences that were measured in DBS specimens were not relevant.     

Ethyl sulfate in blood shows the potential to distinguish alcoholic death and postmortem alcohol instillation

Alcohol is often found in the blood of the deceased. To cover up the true cause of victim’s death, postmortem instillation of alcohol occurs in some criminal cases. Explaining the finding of alcohol is extremely vital in forensic practice. This study aims to evaluate whether ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood and vitreous humor (VH) can be used to distinguish alcoholic death and postmortem alcohol instillation. Saline or 12.6 g/kg ethanol (antemortem alcohol poisoning group) was introduced into rabbits’ stomachs 2 h before sacrificed. Same amount of ethanol was introduced into rabbits’ stomachs at 0 h, 0.5 h, 1 h and 2 h after death in four subgroups of postmortem alcohol instillation group, respectively. Cardiac blood and VH were collected at 10 min, 4 h, 10 h and 24 h after death in blank and antemortem alcohol poisoning group, and after instillation of alcohol in postmortem alcohol instillation group. Blood was also collected at 34 h. Ethanol and EtG levels in blood and VH and EtS in VH in antemortem alcohol poisoning group were overlapped with those in postmortem alcohol instillation group. The contents of EtG and EtS in blood in antemortem alcohol poisoning group (mean ≥ 7.833 μg/mL for EtG and ≥ 19.990 μg/mL for EtS) were much higher than those in postmortem alcohol instillation group (mean ≤ 0.118 μg/mL for EtG and ≤ 0.091 μg/mL for EtS), but apparent decomposition was observed in EtG, which might lead to misinterpretation. Blood EtS showed better stability and could be used to distinguish alcoholic death and postmortem alcohol instillation.     

Alternative matrices in forensic toxicology: a critical review

Forensic Toxicology - The use of alternative matrices in toxicological analyses has been on the rise in clinical and forensic settings. Specimens alternative to blood and urine are useful in...     

Detection of in utero ethanol exposure via ethyl glucuronide and ethyl sulfate analysis in umbilical cord and placenta

Forensic Toxicology - Alcohol exposure during pregnancy constitutes one of the leading preventable causes of birth defects, mental retardation and neurodevelopmental disorders in the exposed...     

Kinetics in serum and urinary excretion of ethyl sulfate and ethyl glucuronide after medium dose ethanol intake

The direct ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), are of increasing importance for clinical and forensic applications, but there are only few studies on the kinetics of EtG in serum and none on EtS. In this study, 13 volunteers (social drinkers) drank ethanol in the form of white wine to reach a blood alcohol concentration of 0.51 ± 0.17 g/kg, and blood and urine samples were analyzed for EtG and EtS simultaneously by chromatography-tandem mass spectrometry (LC-MS/MS). Mean peak serum EtG and EtS concentrations were 2.9 ± 1.3 and 2.8 ± 1.6 μmol/l, respectively, and were reached between 4.0 ± 0.9 h after the start of drinking (3.0 ± 0.5 h for EtS). The mean time differences between reaching maximum blood ethanol levels and serum metabolite levels were 2.3 ± 0.9 h for EtG and 1.2 ± 0.5 h for EtS. In the last blood samples collected (10–11 h after the start of drinking), 11 (of 13) volunteers were still positive for EtG in serum, whereas only 2 were positive for EtS. In the serum of one female person, no EtS was detectable at any time; however, it was excreted in the urine in (low) concentrations. Ethanol was detectable in the serum for up to 8.6 h after the start of drinking, whereas EtG and EtS were detectable up to more than 5.8 h (EtG) and 4.0 h (EtS), respectively. Mean peak urinary concentrations were 401 ± 232 μmol/l for EtG and 266 ± 153 μmol/l for EtS, and mean peak levels were reached 6.2 ± 0.9 h (EtG) and 5.3 ± 1.2 h (EtS) after the start of drinking. Maximum concentrations of EtG and EtS in serum showed a wide interindividual variation and could not be correlated to the maximum blood ethanol concentrations. Correlations (p < 0.001, Kendall’s Tau b) were found when comparing pairs of parameters, but mostly involved areas under the curve (AUC) of metabolites or of ethanol; one correlation linked the peak concentrations of EtG and EtS in urine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ethyl glucuronide concentrations in beard hair after a single alcohol dose: evidence for incorporation in hair root

Background

Despite the growing importance of ethyl glucuronide (EtG) in hair for detection of chronic excessive alcohol consumption, the mechanism of incorporation is not yet clear. Deposition from sweat is believed to be the main route. In order to get more information, EtG was determined in daily shaved beard hair after single higher alcohol doses.

Methods

Three volunteers drank within 5.5?h 153, 165 and 200?g ethanol followed by abstinence. Daily shaved beard hair was analysed for EtG using a validated liquid chromatography–tandem mass spectrometry method with a limit of quantification of 2?pg/mg.

Results

For all three volunteers, small concentrations of EtG were already detected 9?h after end of drinking. The concentrations increased to maxima of 182, 242 and 74?pg/mg on days 2 to 4 and then gradually decreased to limit of quantification on days 8 to 10.

Discussion

The time course of EtG is discussed based on literature data about anatomic dimensions of the hair root, physiology of hair growth, kinetics of EtG formation and elimination in blood, and in comparison to literature results about drugs in beard hair. It follows that for beard hair the predominant portion of EtG is incorporated in the upper part of the hair root between suprabulbar region and isthmus leading to a positive zone of about 3?mm (8–9?days) after a single drinking event. Deposition from sweat which is only possible into the residual hair stubble after shaving and in the infundibulum down to the sebaceous gland mouth was found to be of minor importance but could play a greater role in long hair.

Conclusion

It is concluded that EtG in hair fulfils the prerequisites for time-resolved interpretation of segmental concentrations and that a single excessive drinking can be well detected in sufficiently short hair segments. However, in the routinely investigated 3-cm proximal scalp hair segment and using the cutoff of 7?pg/mg, a negative result can be expected with high probability because of dilution by negative hair.     

Preliminary investigations on ethyl glucuronide and ethyl sulfate cutoffs for detecting alcohol consumption on the basis of an ingestion experiment and on data from withdrawal treatment

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are commonly used alcohol markers for previous alcohol consumption. Nevertheless, the optimum EtG cutoff for urinary abstinence tests is still being discussed, and no cutoff has been recommended for EtS yet. The aim of this study was to verify cutoffs by investigating EtG and EtS concentrations (c(EtG) and c(EtS)) in the urine of healthy persons after drinking small, but realistic amounts of alcohol (one or two glasses of beer or white wine), and to look for the window of detection in strongly alcohol-intoxicated patients who were beginning withdrawal treatment. Very high EtG and EtS concentrations were measured in the first urine samples of patients under withdrawal treatment. However, 24?h later, concentrations decreased considerably, and c (EtG)?相似文献   

Micro-segmental hair analysis: detailed procedures and applications in forensic toxicology

Forensic Toxicology - Since the 1980s, the detection sensitivity of mass spectrometers has increased by improving the analysis of drugs in hair. Accordingly, the number of hair strands required for...     

A novel and an effective analytical approach for the LC-MS determination of ethyl glucuronide and ethyl sulfate in urine

An alternative liquid chromatography–mass spectrometry (LC-MS) method based on no discharge (ND) atmospheric pressure chemical ionization (APCI) was developed for the simultaneous determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in urine in negative ion conditions. Abundant [M-H]^? species of EtG and EtS were obtained, allowing to reach limits of quantification (0.1 µg/ml for both analytes), accuracy, and precision comparable to those proposed in the literature. Additionally, the LC-ND–APCI-MS method proved to be reliable, requiring little maintenance even when high throughput analyses (i.e., 6,000 samples per year) were required.     

Quantification of ethyl glucuronide,ethyl sulfate,nicotine, and its metabolites in human fetal liver and placenta

Purpose

Tobacco and alcohol use during pregnancy is serious public health concerns and result in adverse developmental outcomes. Identifying in utero exposure is often achieved through meconium analysis or via maternal self-report. In this study, we analyzed fetal liver and placenta to examine second trimester alcohol and smoking exposure.

Methods

A validated liquid chromatography–tandem mass spectrometry method for simultaneous analysis of nicotine and its metabolites and alcohol markers (ethyl glucuronide: EtG and ethyl sulfate: EtS) was employed to analyze 193 fetal liver and 48 placenta (n = 47 paired) samples from electively terminated pregnancies.

Results

EtG, EtS, and nicotine markers’ limits of detection were 0.7–20 ng/g in fetal samples. Ninety-eight fetal liver and 23 placenta samples were EtG/EtS-positive, while 137 liver and 25 placenta samples were positive for tobacco exposure. When both alcohol markers were present in samples, EtG/EtS ratios were 1.6–11.1 in 17 livers and 2.5–31.1 in 10 placentas. Median (range) summed tobacco marker concentrations were 422 (1.0–2776) and 154 (1.6–1621) ng/g in livers and placentas, respectively. Median EtG and nicotine marker concentrations were higher in liver than placenta in paired samples. Strong evidence of exposure occurred in 11 and 22 pairs, respectively, when both samples were positive for alcohol and/or tobacco markers.

Conclusions

These paired fetal liver and placenta alcohol and tobacco data provided a unique means for examining the effects of in utero exposure, a critical first step in selecting fetal samples for proteomic and RNA sequencing studies that could provide mechanisms for adverse developmental outcomes.

     

Detecting alcohol abuse: traditional blood alcohol markers compared to ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) measurement in hair

Alcohol abuse is a common problem in society; however, the technical capabilities of evaluating individual alcohol consumption using objective biomarkers are rather limited at present. In recent years research has focused on alcohol markers using hair analysis but data on performance and reliable cut-off values are still lacking. In this study 169 candidates were tested to compare traditional biomarkers, such as carbohydrate-deficient-transferrin (CDT), gamma glutamyl transferase (GGT), aspartate amino transferase, alanine amino transferase and the mean corpuscular volume of the erythrocytes, with alcohol markers detectable in hair such as ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). This study revealed that EtG, GGT and CDT showed the best results, demonstrating areas under the curve calculated from receiver operating characteristics of 0.941, 0.943 and 0.899 respectively. The lowest false-negative and false-positive rates were obtained by using a combined interpretation system for hair EtG and FAEEs. All markers demonstrated only low to moderate correlations. Optimum cut-off values for differentiation between social and chronic excessive drinking calculated for hair EtG and FAEEs were 28 pg/mg and 0.675 ng/mg, respectively. The critical values published in the “Consensus on Alcohol Markers 2012” by the Society of Hair Testing were confirmed.     

Diagnosable and non-diagnosable causes of death by postmortem computed tomography: a review of 339 forensic cases

Postmortem computed tomography (PMCT) is often used to diagnose causes of death, especially in nations with a low autopsy rate. To identify the causes of death that can and cannot be determined by PMCT, imaging findings were reviewed in 339 consecutive forensic autopsy cases. Causes of death could be determined based on PMCT findings alone in 7% of these cases, based on suggestive PMCT findings with additional information in 54%, and could not be determined by PMCT in 38%. PMCT screening may be useful for establishment of some causes of death, including traumatic intracranial hematoma, endogenous intracranial hemorrhage, and some cases of cardiac rupture. Suggestive findings from PMCT in other cases, such as those involving subarachnoid hemorrhage or pericardial hematoma, can lead to misdiagnosis and may be a pitfall of PMCT screening. Causes of death including some cases of cervical cord injuries, asphyxiation, burn, drug intoxication, acute myocardial infarction, and pulmonary thromboembolism cannot be diagnosed using PMCT.     

A fatal clomipramine intoxication case of a chronic alcoholic patient: application of postmortem hair analysis method of clomipramine and ethyl glucuronide using LC/APCI/MS

Toxicological investigations of postmortem specimens of a 26-year-old man were performed with the use of LC/APCI/MS. They revealed in the blood of the deceased clomipramine (9.49 microg/g) and its main metabolite norclomipramine (1.10 microg/g) at concentrations explaining the fatal outcome. The presence of these xenobiotics in a 12-cm-long strand of hair (clomipramine, 7.60 ng/mg in I segment; 4.19 ng/mg in II segment; 1.86 ng/mg in III segment; norclomipramine, 5.71 ng/mg in I segment; 9.71 ng/mg in II segment; 4.13 ng/mg in III segment) confirmed the fact obtained from the medical history that the deceased had been receiving clomipramine as an antidepressant for 1 year prior to his death. The analysis demonstrated ethanol in autopsy blood (2.5mg/ml) and urine (3.2mg/ml); ethyl glucuronide as a marker of chronic alcohol abuse was detected in the deceased's hair (0.44 ng/mg in I segment; 0.07 ng/mg in II segment; n.d. in III segment). These findings may suggest the contribution of alcohol in the mechanism of drug-ethanol interaction, which in consequence might have affected the biotransformation of clomipramine in the final period of his life and evoked the ultimate toxic effect.     

Diagnostic performance of ethyl glucuronide in hair for the investigation of alcohol drinking behavior: a comparison with traditional biomarkers

Background  

Ethyl glucuronide (EtG) in hair has emerged as a useful biomarker for detecting alcohol abuse and monitoring abstinence. However, there is a need to establish a reliable cutoff value for the detection of chronic and excessive alcohol consumption.